The overall goal of this work is to develop anatomical, functional, and molecular magnetic resonance imaging (MRI) techniques that allow non-invasive assessment of brain function and apply these tools to study plasticity and learning in the rodent brain. MRI techniques are having a broad impact on understanding brain. Anatomical based MRI has been very useful for separating gray and white matter and detecting numerous brain disorders. Functional MRI techniques enable detection of regions of the brain that are active during a task. Molecular MRI is an emerging area, whose major goal is to image a large variety of processes in tissues. The goal of this project is to translate MRI developments in all these areas to study system level changes that occur in the rodent brain during plasticity and learning. Aim 1: Progress has been slow in this aim as new fellow learns the challenging MRI techniques required. Over the past few years, we have completed studies in the rodent brain that acquired very high temporal and spatial resolution functional MRI (fMRI) to monitor changes in hemodynamics as a surrogate marker of electrical activity during forepaw stimulation. We have demonstrated that fMRI from single venuoles can be detected with BOLD fMRI and that single arterioles from deep cortex can be effectively imaged using blood volume based MRI techniques. Consistent with our understanding, arteriole blood volume responds earlier that venous BOLD and the two vessel populations can be separated based on time to peak response. In blood volume the arteriole contribution dominates as compared to the tissue or venuole contributions. For BOLD the venuole contribution dominates compared to the arteriole contribution. In related work we have demonstrated that initial BOLD response coincides with the neural input to the cortex. Over the past year we have begun studies to measure the onset distribution through the cortex of arteriole volume and to determine if we can measure the rate of back propagation of arteriole dilation from its origin through the cortical column. In addition, we have begun to quantitatively asses what percent of arterioles and venuoles can be detected by careful comparison of MRI to histology. Aim 2: Over the past several years we have demonstrated that manganese (Mn) chloride enables MRI contrast that defines neural architecture, can monitor activity, can be used to trace neural connections and can be used to monitor neurodegeneration at a cytoarchitectural level. A study is being completed for publication that uses a hippocampal slice preparation and isolated pancreatic beta cells to study the synaptic mechanism underlying the MRI properties of manganese. Results indicate that a number of transport processes influence the accumulation and transport of Mn in these slice cultures. In addition, in collaboration with Richard Leapman we have been able to accomplish very high resolution localization of Mn to synaptic vesicles in neurons and beta cells in frozen tissue helping to validate the model which had hypothesized that Mn is released at synapses. Work is in progress to determine if Mn release can be detected from beta cells. Related to the mechanisms of Mn in movement in the brain we are completing a study quantitating transport of Mn through the Choroid plexus into CSF to determine if this will be useful for measuring function of the choroid plexus Aim 3: Over the past few years we established a rodent model that uses peripheral denervation to study brain plasticity in response to the injury. Over the past couple of years we have shown that denervation of the infraorbital nerve leads to large increases in barrel cortex responses along the spared whisker pathway as well as large ipsilateral cortical activity consistent with our previous work in the forepaw and hindpaw. fMRI and manganese enhanced MRI predicted a strengthening of thalamo-cortical input along the spared pathway which was verified in slice electrophysiology studies in collaboration with John Isaac. Prior to this it was widely believed that the thalamo-cortical input was not capable of strengthening after the critical period. The synaptic mechanisms of this strengthening are caused by a re-activation of the ability of this synapse to demonstrate long term potentiation. This LTP is occurring via activation of silent synapses. Interestingly, LTP is not dependent on the NR2B subunit, but infusion of NR2B inhibitors into the rodent cortex indicate that NR2B is important for establishing silent synapses. We have shown that trimming whiskers has a similar effect as infraorbital nerve cut showing that down regulation of use is enough to cause this LTP in the good whisker pathway. We have begun calcium optical imaging experiments to determine the cellular correlates of this plasticity. Two major questions are: Are more layer 4 stellate neurons firing to the same stimulation?; and, is the relative distribution of S1 output to S2 and M1 altered. We have published a major paper detailing cellular mechanism for takeover by the good whiskers of the denervated whiskers S1 barrel cortex via the corpus callosum input. This input can undergo LTP in the adult and the callosal inputs are strengthened on to layer 5 pyramidal neurons. This strengthening is so large that this synapse can no longer undergo LTP. Inducing LTD in these neurons enables LTP showing that the machinery for LTP is still present. Preliminary data shows interesting differences in presynaptic plasticity depending on which area of the brain that the layer 5 neuron sends outputs, indicating that this plasticity may have specific functional consequences. Finally to begin to get at mechanism for the detected plasticity the group has learned to do single cell RNA seq had has begun to try and understand the cellular state changes that occur. Aim 4: Progress in this aim over the past year has been slow due to lack of a specific person to carry out the work. We have re-established our ability to do fear condition experiments with a new behavioral set-up which was built to be consistent with the new NIMH Behavioral Core behavior enabling easy transfer of skills. We are validating that earlier Mn tracing experiments that indicated plasticity at novel synapses during fear conditioning are reproducible.. If so we will take the approach as Aim 3 to determine the synaptic basis for the changes using slice electrophysiology.